Methoxy-substituted 9-aminomethyl-9,10-dihydroanthracene (AMDA) derivatives exhibit differential binding affinities at the 5-HT2A receptor

Article abstract of DOI:10.1016/j.bmcl.2008.08.059

The effects of methoxy-substitution at the 1-, 2-, 3-, and 4-positions of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on h5-HT_2A receptor affinity were determined. Racemic mixtures of these compounds were found to show the following affinity tr

Full text of DOI:10.1016/j.bmcl.2008.08.059

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5268–5271
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Methoxy-substituted 9-aminomethyl-9,10-dihydroanthracene (AMDA)
derivatives exhibit differential binding affinities at the 5-HT2A receptor
Gajanan K. Dewkar a, Srinivas Peddi a, Philip D. Mosier a, Bryan L. Roth b, Richard B. Westkaemper a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 410 N. 12th Street, PO Box 980540, Richmond, VA 23298-0540, USA
b Departments of Pharmacology and Medicinal Chemistry, Lineberger Cancer Center, Neurosciences Program and NIMH Psychoactive Drug Screening Program,
University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The effects of methoxy-substitution at the 1-, 2-, 3-, and 4-positions of 9-aminomethyl-9,10-dihydroan-
thracene (AMDA) on h5-HT2A receptor affinity were determined. Racemic mixtures of these compounds
were found to show the following affinity trend: 3-MeO > 4-MeO > 1-MeO ꢀ 2-MeO. Comparison of the
effects of these substitutions, with the aid of computational molecular modeling techniques, suggest that
the various positional and stereochemical isomers of the methoxy-substituted AMDA compounds inter-
act differently with the h5-HT2A receptor. It is predicted that for the compounds with higher affinities, the
methoxy oxygen atom is able to interact with hydrogen bond-donating sidechains within alternative h5-
HT2A receptor binding sites, whereas the lower-affinity isomers lack this ability.
Received 27 May 2008
Revised 15 August 2008
Accepted 18 August 2008
Available online 22 August 2008
Keywords:
5-HT2A
AMDA
Homology model
.
Ó 2008 Elsevier Ltd. All rights reserved.
The chemical features that are responsible for the potency and
efficacy of agents that bind to 5-HT2A receptors, as well as the
receptor–ligand interactions responsible for the ligands’ observed
affinities, have not yet been fully elucidated. Toward that end,
we have synthesized and assayed many analogs1 of the selective2
positional isomers, only 3-methoxy-AMDA (4) showed increased
affinity relative to the parent compound AMDA (Ki = 20 nM). 4-
Methoxy-AMDA (5, Ki = 124 nM) displayed a modest sixfold de-
crease in affinity compared to AMDA, while both 1- and 2-meth-
oxy-AMDA (2, Ki = 1158 nM and 3, Ki = 1367 nM, respectively)
were found to have substantially reduced affinity (>50-fold) com-
pared to AMDA.
5-HT2A
antagonist3
9-aminomethyl-9,10-dihydroanthracene
(AMDA, 1). This work in particular builds upon earlier studies1 of
AMDA analogs in which it was shown that substituents of widely
varying size and polarity may be placed at the 3-position without
a large decrease in 5-HT2A affinity. In order to more completely
determine the extent of this apparent steric and/or electrostatic
tolerance, and in particular to find AMDA analogs that have a sig-
nificantly reduced affinity for the 5-HT2A receptor, a series of
AMDA analogs that are substituted at varying positions around
the AMDA core were synthesized and tested. The methoxy group
was chosen a general-purpose ‘probe’ functional group because
of its synthetic flexibility, small size, and amphiphilic nature (i.e.,
it is able to participate in both polar and non-polar interactions).
The results of a systematic study of methoxy-substituted AMDA
compounds, and the concomitant effects that these substitutions
have upon the compounds’ affinity for the human 5-HT2A receptor
are reported here.
Previous receptor modeling studies1 have identified two possi-
ble binding sites for 5-HT2A ligands, as shown schematically in
Figure 1. Site 1 is flanked primarily by transmembrane helices
TM3, TM5, and TM6, and has been proposed as the binding site
for agonists,4,5 partial agonists,6 and antagonists.7,8 Site 2 is an
alternative binding site and is flanked by TM1, TM2, TM3, and
TM7. Ketanserin9, a prototypical 5-HT2A antagonist, and similar
butyrophenones10 have been proposed to simultaneously bind in
both sites.
Each compound in Table 1 (with explicit consideration of ster-
eoisomers) was docked into two graphical receptor models repre-
senting the agonist-selected Site 1 and the antagonist-selected Site
2 (see Experimental methods). The overall quality of the docked
poses was determined using the ChemScore fitness function with
visual inspection of the docked solutions, and the top-ranking solu-
tion for each isomer was selected (see Supplemental Table S1). In
each case, GOLD was able to place the ligand into both Site 1 and
Site 2. However, productive hydrogen bonds were formed only
for the more potent 3-methoxy-AMDA ((R)-4 and (S)-4) and 4-
methoxy-AMDA ((S)-5).11 For (R)-4, the methoxy oxygen H-bonds
with S1593.36, for (S)-4, the H-bond donor is S1312.61, and for
(S)-5, H-bonding occurs with S2395.43 (see Fig. 2). In each of these
Radioligand binding data (5-HT2A receptor affinities) were ob-
tained for each of the target compounds (Table 1). Within the
methoxy-substituted AMDA series, the affinities varied by more
than 180-fold (4, Ki = 7.5 nM; 3, Ki = 1367 nM). Of all four
* Corresponding author. Tel.: +1 804 828 6449; fax: +1 804 828 7625.
E-mail address: rbwestka@vcu.edu (R.B. Westkaemper).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.059

G. K. Dewkar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5268–5271
5269
Table 1
Observed binding affinities for AMDA and methoxy-substituted AMDA analogs at the
h5-HT2A receptor
H2N
R1
8
1
R2
R3
2
7
6
9
3
4
R4
10
5
Compound
R1
R2
R3
R4
Ki, nMa
1
2
3
4
5
–H
–OCH3
–H
–H
–H
–H
–H
–OCH3
–H
–H
–H
–H
–OCH3
–H
–H
–H
–H
–H
20
1158
1367
7.5
–H
–OCH3
124
a
[3H]Ketanserin labeled cloned 5-HT2A sites. Values represent the mean of
computer-derived Ki estimates (using GraphPad Prism) of quadruplicate determi-
nations. Standard errors typically range between 15% and 25% of the Ki value.
Figure 1. Schematic representation of sterically accessible binding sites (Site 1 and
Site 2) within the 5-HT2A receptor.
solutions, the ammonium ion interacts with D1553.32 and with
other lipophilic/aromatic residues surrounding the AMDA core
(see Supplemental Table S2). The current models are consistent
with earlier mutagenesis and molecular modeling studies.12 The
fact that both isomers of 4 are able to H-bond with the receptor
and both receive a relatively high ChemScore is consistent with
its low Ki value. Although there are several hydrogen bond donat-
ing sites within the agonist-selected Site 1 (S1593.36, T1603.37
,
S2395.43, S2425.46, W3366.48, N3436.55) and antagonist-selected Site
2 (S771.35, T811.39, S1312.61, W1513.28, S1593.36, S3737.46), our re-
sults suggest that 1-methoxy AMDA (2) and 2-methoxy-AMDA
(3) are not able to position themselves within these sites in a
way that allows the methoxy group to beneficially interact with
them. This in turn is consistent with the significantly reduced affin-
ity of 2 and 3 for h5-HT2A. The binding affinity of the methoxy-
AMDA compounds can thus be directly correlated with their ability
to H-bond with residues in Site 1 or Site 2.
It should be noted that an alternative explanation for the re-
duced activity of 2 is the possibility of internal H-bond formation.
This would reduce the effectiveness of the amine and the methoxy
group to act as an H-bond donor and acceptor, respectively, and
could potentially impose a ligand conformation that reduces the
complementarity of the ligand to its binding site.
Figure 2. Proposed binding modes of the three MeO-AMDA isomers that exhibit
substantial hydrogen-binding interactions with residues within the binding crevice
of the h5-HT2A receptor. The receptor backbone for models representing Site 1
(agonist-selected) are indicated with a green ribbon; those representing Site 2
(antagonist-selected) are indicated with an orange ribbon. The sidechains of 5-HT2A
residues within 5 Å of the ligand are displayed as capped sticks and the ligand is
rendered as a ball-and-stick model. (a) (R)-3-Methoxy-AMDA ((R)-4) in Site 2. (b)
(S)-3-Methoxy-AMDA ((S)-4) in Site 2. (c) (S)-4-Methoxy-AMDA ((S)-5) in Site 1.
AMDA (1) was synthesized as previously described.3 The syn-
thesis of 1-methoxy- (2) and 2-methoxy-9-aminomethyl-9,10-
dihydroanthracene (3) (Scheme 1) was brought about by the 1,4-
addition of 2-[(methoxymethoxy)-methyl]phenylmagnesium bro-
mide13 to the nitrostyrenes 6a and 6b14 to give substituted 1,1-dia-
ryl-2-nitroethanes 7a and 7b. The MOM-protected benzyl alcohols
7a and 7b were deprotected using HCl to give the nitro alcohols 8a
and 8b, which were reduced to their respective amines to give ami-
no alcohols 9a and 9b. Cyclodehydration of amino alcohols 9a and
9b using freshly prepared PPE in CHCl3 gave the 1- and 2-methoxy
substituted AMDAs 2 and 3, respectively. Synthesis of 3-methoxy-
(4) and 4-methoxy-9-aminomethyl-9,10-dihydroanthracenes (5)
(Scheme 2) was brought about by a standard Suzuki cross-coupling

5270
G. K. Dewkar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5268–5271
NO2
NO2
NO2
R1
R1
R1
R2
R2
R2
a
b
OH
OMOM
6a, b
7a, b
8a, b
NH2
NH2
a R1 = OMe, R2 = H
b R1 = H, R2 = OMe
R1
R1
R2
R2
d
c
2 R1 = OMe, R2 = H
3 R1 = H, R2 = OMe
OH
9a, b
Scheme 1. Reagents and conditions: (a) 1-bromo-2-((methoxymethoxy)methyl)benzene, Mg, I2, THF, 65 ? 0 °C, 12 h; (b) HCl, MeOH, 65 °C, 5 h; (c) H2, (50 psi), MeOH, 10%
Pd/C, 25 °C, 36 h; (d) PPE, CHCl3, reflux, 3 h.
CN
OTMS
CHO
a
b
Br
R2
R2
R2
R1
R1
R1
12a, b
10a, b
11a,b
NH2
NH2
OH
a R1 = H, R2 = OMe
b R1 = OMe, R2 = H
d
c
R2
R2
R1
R1
13a,b
4 R1 = H, R2 = OMe
5 R1 = OMe, R2 = H
Scheme 2. Reagents and conditions: (a) 2-formylphenylboronic acid, Pd(PPh3)4, Na2CO3, toluene/ethanol (9:1), 100 °C, 3 h; (b) TMSCN, ZnI2, CH2Cl2, 65 °C, 4 h; (c) LiAlH4,
THF, reflux, 12h; (d) CH3SO3H, 25 °C, 12h; PPA, 60 °C, 6 h.
reaction between commercially available 3-methoxybenzyl bro-
mide 10a or 2-methoxybenzyl bromide 10b15 and 2-form-
ylphenylboronic acid to yield 2-(3-methoxybenzyl)benzaldehyde
11a and 2-(2-methoxybenzyl)benzaldehyde 11b. Cyanosilylation
of the aldehydes 11a and 11b using TMSCN gave the corresponding
cyano trimethylsilyl ethers 12a and 12b, which were reduced with
LiAlH4 to give the respective amino alcohols 13a and 13b. Cyclode-
hydration of amino alcohols 13a and 13b using CH3SO3H and PPA,
respectively, gave the 3-methoxy- and 4-methoxy-substituted AM-
DAs 4 and 5.
The molecular modeling methodology used to generate the h5-
HT2A receptor–ligand complex models will be described briefly
here, and is discussed in detail elsewhere.12 To begin, the h5-
HT2A receptor sequence was aligned using ClustalX 1.8316 with a
profile of several related class A GPCRs17 that included bovine rho-
dopsin. The result was an unambiguous alignment (in the TM heli-
cal regions) of the h5-HT2A sequence with that of bovine
rhodopsin. Manual modifications were made to the alignment in
the region of the second extracellular loop to properly align the
cysteine residues of the disulfide linkage. This alignment, along
with a file containing the atomic coordinates of bovine rhodopsin
(‘A’ chain of PDB code 1U19), was used as input to the MODELLER
software package18,19 to generate a population of 100 h5-HT2A
homology models, each with a different conformation. For the tem-
plate rhodopsin structure, all residues within 12 Å of the bound
retinal ligand were mutated to alanine to encourage MODELLER
to produce structurally diverse receptor conformations. The N-
and C-termini were truncated. The third intracellular loop was
modeled simply as a poly-Gly chain whose backbone coordinates
were taken from the structure of rhodopsin. Each receptor in the
population was subsequently energy-minimized without con-
straint in SYBYL 7.2 (Tripos Inc., St. Louis, MO) using the Tripos
Force Field (TFF) with Gasteiger–Hückel charges, a distance-depen-
dent dielectric constant of 4, a non-bonded interaction cutoff = 8 Å,
and were terminated at an energy gradient of 0.05 kcal/(mol Å).
The automated docking program GOLD20,21 version 3.01 (Cam-
bridge Crystallographic Data Centre, Cambridge, UK) was then used
to dock a potent 5-HT2A agonist (1-(2,5-dimethoxy-4-bromophe-
nyl)-2-aminopropane; DOB) and a potent antagonist (ketanserin)
into each of the 100 receptor models. Based on the fitness scores
and quality of the docked poses, one receptor model was selected
to represent the agonist binding site (Site 1 in Fig. 1) and a second
model was selected to represent an additional antagonist binding
site (Site 2 in Fig. 1). After a small amount of conformational refine-
ment and checks for stereochemical integrity, the two receptor
models were saved and used for subsequent docking exercises.
Ligand molecules were sketched in using SYBYL and energy-
minimized using the same method and parameters as were used
for the receptor models. Basic amines were protonated to form
ammonium ions. Ligand chirality was treated explicitly, with each
isomer sketched, energy-minimized and saved as a separate struc-
ture file. GOLD was then used to dock each ligand structure into
each of the two selected receptor models. The parameter set de-
fined by the ‘standard default settings’ option was used in conjunc-
tion with a protein H-bond constraint (default settings) that biased
the docked solutions in favor of those in which the ligand ammo-
nium ion interacted with the conserved D1553.32. Ten genetic algo-
rithm (GA) runs were performed for each ligand at both Site 1 and
Site 2. Short molecular dynamics (MD) simulations were then car-
ried out to enable the receptor–ligand complex to sample alterna-

G. K. Dewkar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5268–5271
5271
tive locally accessible low-energy states in order to improve the
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This work was supported by United States Public Health Service
Grant R01-MH57969 (RBW), R01-MH57635 (BLR), K02-MH01366
(BLR) and R01-MH61887 (BLR), U19-MH82441 (BLR) and the
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.059.